Lipid nanoparticle compositions and methods for mrna delivery

ABSTRACT

Disclosed herein are compositions and methods for modulating the production of a protein in a target cell. The compositions and methods disclosed herein are capable of ameliorating diseases associated with protein or enzyme deficiencies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 16/502,672 filed Jul. 3, 2019, now allowed, which is adivisional application of U.S. application Ser. No. 16/286,400 filed onFeb. 26, 2019, issued as U.S. Pat. No. 10,350,303 on Jul. 16, 2019,which is a divisional application of U.S. application Ser. No.16/233,031 filed on Dec. 26, 2018, now issued as U.S. Pat. No.10,413,618, which is a divisional application of U.S. application Ser.No. 15/482,117 filed on Apr. 7, 2017, now issued as U.S. Pat. No.10,238,754, which is a divisional application of U.S. application Ser.No. 14/124,608 filed on Mar. 5, 2014, now abandoned, which is a U.S.National Entry claiming priority to International ApplicationPCT/US2012/041724 filed on Jun. 8, 2012, which claims priority to U.S.Provisional Application Ser. No. 61/494,881 filed Jun. 8, 2011, thedisclosures of which are hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

This instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 12, 2019, isnamed MRT-1055US8_ST25.txt and is 10,122 bytes in size. No new matter ishereby added.

Novel approaches and therapies are still needed for the treatment ofprotein and enzyme deficiencies. For example, lysosomal storage diseasesare a group of approximately 50 rare inherited metabolic disorders thatresult from defects in lysosomal function, usually due to a deficiencyof an enzyme required for metabolism. Fabry disease is a lysosomalstorage disease that results from a deficiency of the enzyme alphagalactosidase (GLA), which causes a glycolipid known asglobotriaosylceramide to accumulate in blood vessels and other tissues,leading to various painful manifestations. For certain diseases, likeFabry disease, there is a need for replacement of a protein or enzymethat is normally secreted by cells into the blood stream. Therapies,such as gene therapy, that increase the level or production of anaffected protein or enzyme could provide a treatment or even a cure forsuch disorders. However, there have been several limitations to usingconventional gene therapy for this purpose.

Conventional gene therapy involves the use of DNA for insertion ofdesired genetic information into host cells. The DNA introduced into thecell is usually integrated to a certain extent into the genome of one ormore transfected cells, allowing for long-lasting action of theintroduced genetic material in the host. While there may be substantialbenefits to such sustained action, integration of exogenous DNA into ahost genome may also have many deleterious effects. For example, it ispossible that the introduced DNA will be inserted into an intact gene,resulting in a mutation which impedes or even totally eliminates thefunction of the endogenous gene. Thus, gene therapy with DNA may resultin the impairment of a vital genetic function in the treated host, suchas e.g., elimination or deleteriously reduced production of an essentialenzyme or interruption of a gene critical for the regulation of cellgrowth, resulting in unregulated or cancerous cell proliferation. Inaddition, with conventional DNA based gene therapy it is necessary foreffective expression of the desired gene product to include a strongpromoter sequence, which again may lead to undesirable changes in theregulation of normal gene expression in the cell. It is also possiblethat the DNA based genetic material will result in the induction ofundesired anti-DNA antibodies, which in turn, may trigger a possiblyfatal immune response. Gene therapy approaches using viral vectors canalso result in an adverse immune response. In some circumstances, theviral vector may even integrate into the host genome. In addition,production of clinical grade viral vectors is also expensive and timeconsuming. Targeting delivery of the introduced genetic material usingviral vectors can also be difficult to control. Thus, while DNA basedgene therapy has been evaluated for delivery of secreted proteins usingviral vectors (U.S. Pat. No. 6,066,626; US2004/0110709), theseapproaches may be limited for these various reasons.

Another obstacle apparent in these prior approaches at delivery ofnucleic acids encoding secreted proteins, is in the levels of proteinthat are ultimately produced. It is difficult to achieve significantlevels of the desired protein in the blood, and the amounts are notsustained over time. For example, the amount of protein produced bynucleic acid delivery does not reach normal physiological levels. Seee.g., US2004/0110709.

In contrast to DNA, the use of RNA as a gene therapy agent issubstantially safer because (1) RNA does not involve the risk of beingstably integrated into the genome of the transfected cell, thuseliminating the concern that the introduced genetic material willdisrupt the normal functioning of an essential gene, or cause a mutationthat results in deleterious or oncogenic effects; (2) extraneouspromoter sequences are not required for effective translation of theencoded protein, again avoiding possible deleterious side effects; (3)in contrast to plasmid DNA (pDNA), messenger RNA (mRNA) is devoid ofimmunogenic CpG motifs so that anti-RNA antibodies are not generated;and (4) any deleterious effects that do result from mRNA based on genetherapy would be of limited duration due to the relatively shorthalf-life of RNA. In addition, it is not necessary for mRNA to enter thenucleus to perform its function, while DNA must overcome this majorbarrier.

One reason that mRNA based gene therapy has not been used more in thepast is that mRNA is far less stable than DNA, especially when itreaches the cytoplasm of a cell and is exposed to degrading enzymes. Thepresence of a hydroxyl group on the second carbon of the sugar moiety inmRNA causes steric hinderance that prevents the mRNA from forming themore stable double helix structure of DNA and thus makes the mRNA moreprone to hydrolytic degradation. As a result, until recently, it waswidely believed that mRNA was too labile to withstand transfectionprotocols. Advances in RNA stabilizing modifications have sparked moreinterest in the use of mRNA in place of plasmid DNA in gene therapy.Certain delivery vehicles, such as cationic lipid or polymer deliveryvehicles may also help protect the transfected mRNA from endogenousRNases. Yet, in spite of increased stability of modified mRNA, deliveryof mRNA to cells in vivo in a manner allowing for therapuetic levels ofprotein production is still a challenge, particularly for mRNA encodingfull length proteins. While delivery of mRNA encoding secreted proteinshas been contemplated (US2009/0286852), the levels of a full lengthsecreted protein that would actually be produced via in vivo mRNAdelivery are not known and there is not a reason to expect the levelswould exceed those observed with DNA based gene therapy.

To date, significant progress using mRNA gene therapy has only been madein applications for which low levels of translation has not been alimiting factor, such as immunization with mRNA encoding antigens.Clinical trials involving vaccination against tumor antigens byintradermal injection of naked or protamine-complexed mRNA havedemonstrated feasibility, lack of toxicity, and promising results. X. Suet al., Mol. Pharmaceutics 8:774-787 (2011). Unfortunately, low levelsof translation has greatly restricted the exploitation of mRNA basedgene therapy in other applications which require higher levels ofsustained expression of the mRNA encoded protein to exert a biologicalor therapeutic effect.

The invention provides methods for delivery of mRNA gene therapeuticagents that lead to the production of therapeutically effective levelsof secreted proteins via a “depot effect.” In embodiments of theinvention, mRNA encoding a secreted protein is loaded in lipidnanoparticles and delivered to target cells in vivo. Target cells thenact as a depot source for production of soluble, secreted protein intothe circulatory system at therapeutic levels. In some embodiments, thelevels of secreted protein produced are above normal physiologicallevels.

The invention provides compositions and methods for intracellulardelivery of mRNA in a liposomal transfer vehicle to one or more targetcells for production of therapeutic levels of secreted functionalprotein.

The compositions and methods of the invention are useful in themanagement and treatment of a large number of diseases, in particulardiseases which result from protein and/or enzyme deficiencies, whereinthe protein or enzyme is normally secreted. Individuals suffering fromsuch diseases may have underlying genetic defects that lead to thecompromised expression of a protein or enzyme, including, for example,the non-synthesis of the secreted protein, the reduced synthesis of thesecreted protein, or synthesis of a secreted protein lacking or havingdiminished biological activity. In particular, the methods andcompositions of the invention are useful for the treatment of lysosomalstorage disorders and/or the urea cycle metabolic disorders that occuras a result of one or more defects in the biosynthesis of secretedenzymes involved in the urea cycle.

The compositions of the invention comprise an mRNA, a transfer vehicleand, optionally, an agent to facilitate contact with, and subsequenttransfection of a target cell. The mRNA can encode a clinically usefulsecreted protein. For example, the mRNA may encode a functional secretedurea cycle enzyme or a secreted enzyme implicated in lysosomal storagedisorders. The mRNA can encode, for example, erythropoietin (e.g., humanEPO) or a-galactosidase (e.g., human α-galactosidase (human GLA).

In some embodiments the mRNA can comprise one or more modifications thatconfer stability to the mRNA (e.g., compared to a wild-type or nativeversion of the mRNA) and may also comprise one or more modificationsrelative to the wild-type which correct a defect implicated in theassociated aberrant expression of the protein. For example, the nucleicacids of the present invention may comprise modifications to one or bothof the 5′ and 3′ untranslated regions. Such modifications may include,but are not limited to, the inclusion of a partial sequence of acytomegalovirus (CMV) immediate-early 1 (IE1) gene, a poly A tail, aCap1 structure or a sequence encoding human growth hormone (hGH)). Insome embodiments, the mRNA is modified to decrease mRNA immunogenecity.

Methods of treating a subject comprising administering a composition ofthe invention, are also contemplated. For example, methods of treatingor preventing conditions in which production of a particular secretedprotein and/or utilization of a particular secreted protein isinadequate or compromised are provided. In one embodiment, the methodsprovided herein can be used to treat a subject having a deficiency inone or more urea cycle enzymes or in one or more enzymes deficient in alysosomal storage disorder.

In a preferred embodiment, the mRNA in the compositions of the inventionis formulated in a liposomal transfer vehicle to facilitate delivery tothe target cell. Contemplated transfer vehicles may comprise one or morecationic lipids, non-cationic lipids, and/or PEG-modified lipids. Forexample, the transfer vehicle may comprise at least one of the followingcationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, orHGT5001. In embodiments, the transfer vehicle comprises cholesterol(chol) and/or a PEG-modified lipid. In some embodiments, the transfervehicles comprises DMG-PEG2K. In certain embodiments, the tranfervehicle comprises one of the following lipid formulations: C12-200,DOPE, chol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000,DOPE, chol, DMG-PEG2K, HGT5001, DOPE, chol, DMG-PEG2K.

The invention also provides compositions and methods useful forfacilitating the transfection and delivery of one or more mRNA moleculesto target cells capable of exhibiting the “depot effect.” For example,the compositions and methods of the present invention contemplate theuse of targeting ligands capable of enhancing the affinity of thecomposition to one or more target cells. In one embodiment, thetargeting ligand is apolipoprotein-B or apolipoprotein-E andcorresponding target cells express low-density lipoprotein receptors,thereby facilitating recognition of the targeting ligand. The methodsand compositions of the present invention may be used to preferentiallytarget a vast number of target cells. For example, contemplated targetcells include, but are not limited to, hepatocytes, epithelial cells,hematopoietic cells, epithelial cells, endothelial cells, lung cells,bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells,adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletalmuscle cells, beta cells, pituitary cells, synovial lining cells,ovarian cells, testicular cells, fibroblasts, B cells, T cells,reticulocytes, leukocytes, granulocytes and tumor cells.

In embodiments, the secreted protein is produced by the target cell forsustained amounts of time. For example, the secreted protein may beproducted for more than one hour, more than four, more than six, morethan 12, more than 24, more than 48 hours, or more than 72 hours afteradministration. In some embodiments the polypeptide is expressed at apeak level about six hours after administration. In some embodiments theexpression of the polypeptide is sustained at least at a therapeuticlevel. In some embodiments the polypeptide is expressed at at least atherapeutic level for more than one, more than four, more than six, morethan 12, more than 24, more than 48 hours, or more than 72 hours afteradministration. In some embodiments the polypeptide is detectable at thelevel in patient serum or tissue (e.g., liver, or lung). In someembodiments, the level of detectable polypeptide is from continuousexpession from the mRNA composition over periods of time of more thanone, more than four, more than six, more than 12, more than 24, morethan 48 hours, or more than 72 hours after administration.

In certain embodiments, the secreted protein is produced at levels abovenormal physiological levels. The level of secreted protein may beincreased as compared to a control.

In some embodiments the control is the baseline physiological level ofthe polypeptide in a normal individual or in a population of normalindividuals. In other embodiments the control is the baselinephysiological level of the polypeptide in an individual having adeficiency in the relevant protein or polypeptide or in a population ofindividuals having a deficiency in the relevant protein or polypeptide.In some embodiments the control can be the normal level of the relevantprotein or polypeptide in the individual to whom the composition isadministered. In other embodiments the control is the expression levelof the polypeptide upon other therapeutic intervention, e.g., upondirect injection of the corresponding polypeptide, at one or morecomparable time points.

In certain embodiments the polypeptide is expressed by the target cellat a level which is at least 1.5-fold, at least 2-fold, at least 5-fold,at least 10-fold, at least 20-fold, 30-fold, at least 100-fold, at least500-fold, at least 5000-fold, at least 50,000-fold or at least100,000-fold greater than a control. In some embodiments, the foldincrease of expression greater than control is sustained for more thanone, more than four, more than six, more than 12, more than 24, or morethan 48 hours, or more than 72 hours after administration. For example,in one embodiment,the levels of secreted protein are detected in theserum at least 1.5-fold, at least 2-fold, at least 5-fold, at least10-fold, at least 20-fold, 30-fold, at least 100-fold, at least500-fold, at least 5000-fold, at least 50,000-fold or at least100,000-fold greater than a control for at least 48 hours or 2 days. Incertain embodiments, the levels of secreted protein are detectable at 3days, 4 days, 5 days, or 1 week or more after administration. Increasedlevels of secreted protein may be observed in the serum and/or in atissue (e.g. liver, lung).

In some embodiments, the method yields a sustained circulation half-lifeof the desired secreted protein. For example, the secreted protein maybe detected for hours or days longer than the half-life observed viasubcutaneous injection of the secreted protein. In embodiments, thehalf-life of the secreted protein is sustained for more than 1 day, 2days, 3 days, 4 days, 5 days, or 1 week or more.

In some embodiments administration comprises a single or repeated doses.In certain embodiments, the dose is administered intravenously, or bypulmonary delivery.

The polypeptide can be, for example, one or more of erythropoietin,α-galactosidase, LDL receptor, Factor VIII, Factor IX, α-L-iduronidase(for MPS I), iduronate sulfatase (for MPS II), heparin-N-sulfatase (forMPS IIIA), α-N-acetylglucosaminidase (for MPS IIIB), galactose6-sultatase (for MPS IVA), lysosomal acid lipase, arylsulfatase-A.

Certain embodiments relate to compositions and methods that provide to acell or subject mRNA, at least a part of which encodes a functionalprotein, in an amount that is substantially less that the amount ofcorresponding functional protein generated from that mRNA. Put anotherway, in certain embodiments the mRNA delivered to the cell can producean amount of protein that is substantially greater than the amount ofmRNA delivered to the cell. For example, in a given amount of time, forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 24 hours fromadministration of the mRNA to a cell or subject, the amount ofcorresponding protein generated by that mRNA can be at least 1.5, 2, 3,5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 400, 500, or more timesgreater that the amount of mRNA actually administered to the cell orsubject. This can be measured on a mass-by-mass basis, on a mole-by-molebasis, and/or on a molecule-by-molecule basis. The protein can bemeasured in various ways. For example, for a cell, the measured proteincan be measured as intracellular protein, extracellular protein, or acombination of the two. For a subject, the measured protein can beprotein measured in serum; in a specific tissue or tissues such as theliver, kidney, heart, or brain; in a specific cell type such as one ofthe various cell types of the liver or brain; or in any combination ofserum, tissue, and/or cell type. Moreover, a baseline amount ofendogenous protein can be measured in the cell or subject prior toadministration of the mRNA and then subtracted from the protein measuredafter administration of the mRNA to yield the amount of correspondingprotein generated from the mRNA. In this way, the mRNA can provide areservoir or depot source of a large amount of therapeutic material tothe cell or subject, for example, as compared to amount of mRNAdelivered to the cell or subject. The depot source can act as acontinuous source for polypeptide expression from the mRNA oversustained periods of time.

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples. The various embodimentsdescribed herein are complimentary and can be combined or used togetherin a manner understood by the skilled person in view of the teachingscontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a 5′ CMV sequence (SEQ ID NO:1),wherein X, if present is GGA.

FIG. 2 shows the nucleotide sequence of a 3′ hGH sequence (SEQ ID NO:2).

FIG. 3 shows the nucleotide sequence of human erythropoietic (EPO) mRNA(SEQ ID NO:3). This sequence can be flanked on the 5′ end with SEQ IDNO:1 and on the 3′ end with SEQ ID NO:2.

FIG. 4 shows the nucleotide sequence of human alpha-galactosidase (GLA)mRNA (SEQ ID NO:4). This sequence can be flanked on the 5′ end with SEQID NO:1 and on the 3′ end with SEQ ID NO:2.

FIG. 5 shows the nucleotide sequence of human alpha-1 antitrypsin (A1AT)mRNA (SEQ ID NO:5). This sequence can be flanked on the 5′ end with SEQID NO:1 and on the 3′ end with SEQ ID NO:2.

FIG. 6 shows the nucleotide sequence of human factor IX (FIX) mRNA (SEQID NO:6). This sequence can be flanked on the 5′ end with SEQ ID NO:1and on the 3′ end with SEQ ID NO:2.

FIG. 7 shows quantification of secreted hEPO protein levels as measuredvia ELISA. The protein detected is a result of its production from hEPOmRNA delivered intravenously via a single dose of various lipidnanoparticle formulations. The formulations C12-200 (30 ug), HGT4003(150 ug), ICE (100 ug), DODAP (200 ug) are represented as thecationic/ionizable lipid component of each test article (Formulations1-4). Values are based on blood sample four hours post-administration.

FIG. 8 shows the hematocrit measurement of mice treated with a single IVdose of human EPO mRNA-loaded lipid nanoparticles (Formulations 1-4).Whole blood samples were taken at 4 hr (Day 1), 24 hr (Day 2), 4 days, 7days, and 10 days post-administration.

FIG. 9 shows hematocrit measurements of mice treated with humanEPO-mRNA-loaded lipid nanoparticles with either a single IV dose orthree injections (day 1, day 3, day 5). Whole blood samples were takenprior to injection (day −4), day 7, and day 15. Formulation 1 wasadministered: (30 ug, single dose) or (3×10 ug, dose day 1, day 3, day5); Formulation 2 was administered: (3×50 ug, dose day 1, day 3, day 5).

FIG. 10 shows quantification of secreted human α-galactosidase (hGLA)protein levels as measured via ELISA. The protein detected is a resultof the production from hGLA mRNA delivered via lipid nanoparticles(Formulation 1; 30 ug single intravenous dose, based on encapsulatedmRNA). hGLA protein is detected through 48 hours.

FIG. 11 shows hGLA activity in serum. hGLA activity was measured usingsubstrate 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-gal) at 37°C. Data are average of 6 to 9 individual measurements.

FIG. 12 shows quantification of hGLA protein levels in serum as measuredvia ELISA. Protein is produced from hGLA mRNA delivered viaC12-200-based lipid nanoparticles (C12-200:DOPE:Chol:DMGPEG2K,40:30:25:5 (Formulation 1); 30 ug mRNA based on encapsulated mRNA,single IV dose). hGLA protein is monitored through 72 hours. per singleintravenous dose, based on encapsulated mRNA). hGLA protein is monitoredthrough 72 hours.

FIG. 13 shows quantification of hGLA protein levels in liver, kidney,and spleen as measured via ELISA. Protein is produced from hGLA mRNAdelivered via C12-200-based lipid nanoparticles (Formulation 1; 30 ugmRNA based on encapsulated mRNA, single IV dose). hGLA protein ismonitored through 72 hours.

FIG. 14A shows a dose response study monitoring protein production ofhGLA as secreted MRT-derived human GLA protein in serum. Samples weremeasured 24 hours post-administration (Formulation 1; single dose, IV,N=4 mice/group) and quantified via ELISA.

FIG. 14B shows a dose response study monitoring protein production ofhGLA as secreted MRT-derived human GLA protein in liver. Samples weremeasured 24 hours post-administration (Formulation 1; single dose, IV,N=4 mice/group) and quantified via ELISA.

FIG. 15 shows the pharmacokinetic profiles of ERT-basedAlpha-galactosidase in athymjic nude mice (40 ug/kg dose) and hGLAprotein produced from MRT (Formulation 1; 1.0 mg/kg mRNA dose).

FIG. 16 shows the quantification of secreted hGLA protein levels inMRT-treated Fabry mice as measured using ELISA. hGLA protein is producedfrom hGLA mRNA delivered via C12-200-based lipid nanoparticles(Formulation 1; 10 ug mRNA per single intravenous dose, based onencapsulated mRNA). Serum is monitored through 72 hours.

FIG. 17 shows the quantification of hGLA protein levels in liver,kidney, spleen, and heart of MRT-treated Fabry KO mice as measured viaELISA. Protein is produced from hGLA mRNA delivered via C12-200-basedlipid nanoparticles (Formulation 1; 30 ug mRNA based on encapsulatedmRNA, single IV dose). hGLA protein is monitored through 72 hours.Literature values representing normal physiological levels are graphedas dashed lines.

FIG. 18 shows the quantification of secreted hGLA protein levels in MRTand Alpha-galactosidase-treated Fabry mice as measured using ELISA. Boththerapies were dosed as a single 1.0 mg/kg intravenous dose.

FIG. 19 shows the quantification of hGLA protein levels in liver,kidney, spleen, and heart of MRT and ERT (Alpha-galactosidase)-treatedFabry KO mice as measured via ELISA. Protein produced from hGLA mRNAdelivered via lipid nanoparticles (Formulation 1; 1.0 mg/kg mRNA basedon encapsulated mRNA, single IV dose).

FIG. 20 shows the relative quantification of globotrioasylceramide (Gb3)and lyso-Gb3 in the kidneys of treated and untreated mice. Male Fabry KOmice were treated with a single dose either GLA mRNA-loaded lipidnanoparticles or Alpha-galactosidase at 1.0 mg/kg. Amounts reflectquantity of Gb3/lyso-Gb3 one week post-administration.

FIG. 21 shows the relative quantification of globotrioasylceramide (Gb3)and lyso-Gb3 in the heart of treated and untreated mice. Male Fabry KOmice were treated with a single dose either GLA mRNA-loaded lipidnanoparticles or Alpha-galactosidase at 1.0 mg/kg. Amounts reflectquantity of Gb3/lyso-Gb3 one week post-administration.

FIG. 22 shows a dose response study monitoring protein production of GLAas secreted MRT-derived human GLA protein in serum. Samples weremeasured 24 hours post-administration (single dose, IV, N=4 mice/group)of either HGT4003 (Formulator 3) or HGT5000-based lipid nanoparticles(Formulation 5) and quantified via ELISA.

FIG. 23A shows hGLA protein production as measured in serum. Sampleswere measured 6 hours and 24 hours post-administration (single dose, IV,N=4 mice/group) of HGT5001-based lipid nanoparticles (Formulation 6) andquantified via ELISA.

FIG. 23B shows hGLA protein production as measured in liver, kidney, andspleen (B). Samples were measured 6 hours and 24 hourspost-administration (single dose, IV, N=4 mice/group) of HGT5001-basedlipid nanoparticles (Formulation 6) and quantified via ELISA.

FIG. 24 shows the quantification of secreted human Factor IX proteinlevels measured using ELISA (mean ng/mL±standard deviation). FIX proteinis produced from FIX mRNA delivered via C12-200-based lipidnanoparticles (C12-200:DOPE:Chol:DMGPEG2K, 40:30:25:5 (Formulation 1);30 ug mRNA per single intravenous dose, based on encapsulated mRNA). FIXprotein is monitored through 72 hours. (n=24 mice)

FIG. 25 shows the quantification of secreted human α-1-antitrypsin(A1AT) protein levels measured using ELISA. A1AT protein is producedfrom A1AT mRNA delivered via C12-200-based lipid nanoparticles(C12-200:DOPE:Chol:DMGPEG2K, 40:30:25:5 (Formulation 1); 30 ug mRNA persingle intravenous dose, based on encapsulated mRNA). A1AT protein ismonitored through 24 hours.

FIG. 26 shows an ELISA-based quantification of hEPO protein detected inthe lungs and serum of treated mice after intratracheal administrationof hEPO mRNA-loaded nanoparticles (measured mIU) (C12-200, HGT5000, orHGT5001-based lipid nanoparticles; Formulations 1, 5, 6 respectively).Animals were sacrificed 6 hours post-administration (n=4 mice pergroup).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The invention provides compositions and methods for intracellulardelivery of mRNA in a liposomal transfer vehicle to one or more targetcells for production of therapeutic levels of secreted functionalprotein.

The term “functional,” as used herein to qualify a protein or enzyme,means that the protein or enzyme has biological activity, oralternatively is able to perform the same, or a similar function as thenative or normally-functioning protein or enzyme. The mRNA compositionsof the invention are useful for the treatment of a various metabolic orgenetic disorders, and in particular those genetic or metabolicdisorders which involve the non-expression, misexpression or deficiencyof a protein or enzyme. The term “therapeutic levels” refers to levelsof protein detected in the blood or tissues that are above controllevels, wherein the control may be normal physiological levels, or thelevels in the subject prior to administration of the mRNA composition.The term “secreted” refers to protein that is detected outside thetarget cell, in extracellular space. The protein may be detected in theblood or in tissues. In the context of the present invention the term“produced” is used in its broadest sense to refer the translation of atleast one mRNA into a protein or enzyme. As provided herein, thecompositions include a transfer vehicle. As used herein, the term“transfer vehicle” includes any of the standard pharmaceutical carriers,diluents, excipients and the like which are generally intended for usein connection with the administration of biologically active agents,including nucleic acids. The compositions and in particular the transfervehicles described herein are capable of delivering mRNA to the targetcell. In embodiments, the transfer vehicle is a lipid nanoparticle.

mRNA

The mRNA in the compositions of the invention may encode, for example, asecreted hormone, enzyme, receptor, polypeptide, peptide or otherprotein of interest that is normally secreted. In one embodiment of theinvention, the mRNA may optionally have chemical or biologicalmodifications which, for example, improve the stability and/or half-lifeof such mRNA or which improve or otherwise facilitate proteinproduction.

The methods of the invention provide for optional co-delivery of one ormore unique mRNA to target cells, for example, by combining two uniquemRNAs into a single transfer vehicle. In one embodiment of the presentinvention, a therapeutic first mRNA, and a therapeutic second mRNA, maybe formulated in a single transfer vehicle and administered. The presentinvention also contemplates co-delivery and/or co-administration of atherapeutic first mRNA and a second nucleic acid to facilitate and/orenhance the function or delivery of the therapeutic first mRNA. Forexample, such a second nucleic acid (e.g., exogenous or synthetic mRNA)may encode a membrane transporter protein that upon expression (e.g.,translation of the exogenous or synthetic mRNA) facilitates the deliveryor enhances the biological activity of the first mRNA. Alternatively,the therapeutic first mRNA may be administered with a second nucleicacid that functions as a “chaperone” for example, to direct the foldingof either the therapeutic first mRNA.

The methods of the invention also provide for the delivery of one ormore therapeutic nucleic acids to treat a single disorder or deficiency,wherein each such therapeutic nucleic acid functions by a differentmechanism of action. For example, the compositions of the presentinvention may comprise a therapeutic first mRNA which, for example, isadministered to correct an endogenous protein or enzyme deficiency, andwhich is accompanied by a second nucleic acid, which is administered todeactivate or “knock-down” a malfunctioning endogenous nucleic acid andits protein or enzyme product. Such “second” nucleic acids may encode,for example mRNA or siRNA.

Upon transfection, a natural mRNA in the compositions of the inventionmay decay with a half-life of between 30 minutes and several days. ThemRNA in the compositions of the invention preferably retain at leastsome ability to be translated, thereby producing a functional secretedprotein or enzyme. Accordingly, the invention provides compositionscomprising and methods of administering a stabilized mRNA. In someembodiments of the invention, the activity of the mRNA is prolonged overan extended period of time. For example, the activity of the mRNA may beprolonged such that the compositions of the present invention areadministered to a subject on a semi-weekly or bi-weekly basis, or morepreferably on a monthly, bi-monthly, quarterly or an annual basis. Theextended or prolonged activity of the mRNAof the present invention, isdirectly related to the quantity of secreted functional protein orenzyme produced from such mRNA. Similarly, the activity of thecompositions of the present invention may be further extended orprolonged by modifications made to improve or enhance translation of themRNA. Furthermore, the quantity of functional protein or enzyme producedby the target cell is a function of the quantity of mRNA delivered tothe target cells and the stability of such mRNA. To the extent that thestability of the mRNA of the present invention may be improved orenhanced, the half-life, the activity of the produced secreted proteinor enzyme and the dosing frequency of the composition may be furtherextended.

Accordingly, in some embodiments, the mRNA in the compositions of theinvention comprise at least one modification which confers increased orenhanced stability to the nucleic acid, including, for example, improvedresistance to nuclease digestion in vivo. As used herein, the terms“modification” and “modified” as such terms relate to the nucleic acidsprovided herein, include at least one alteration which preferablyenhances stability and renders the mRNA more stable (e.g., resistant tonuclease digestion) than the wild-type or naturally occurring version ofthe mRNA. As used herein, the terms “stable” and “stability” as suchterms relate to the nucleic acids of the present invention, andparticularly with respect to the mRNA, refer to increased or enhancedresistance to degradation by, for example nucleases (i.e., endonucleasesor exonucleases) which are normally capable of degrading such mRNA.Increased stability can include, for example, less sensitivity tohydrolysis or other destruction by endogenous enzymes (e.g.,endonucleases or exonucleases) or conditions within the target cell ortissue, thereby increasing or enhancing the residence of such mRNA inthe target cell, tissue, subject and/or cytoplasm. The stabilized mRNAmolecules provided herein demonstrate longer half-lives relative totheir naturally occurring, unmodified counterparts (e.g. the wild-typeversion of the mRNA). Also contemplated by the terms “modification” and“modified” as such terms related to the mRNA of the present inventionare alterations which improve or enhance translation of mRNA nucleicacids, including for example, the inclusion of sequences which functionin the initiation of protein translation (e.g., the Kozac consensussequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

In some embodiments, the mRNA of the invention have undergone a chemicalor biological modification to render them more stable. Exemplarymodifications to an mRNA include the depletion of a base (e.g., bydeletion or by the substitution of one nucleotide for another) ormodification of a base, for example, the chemical modification of abase. The phrase “chemical modifications” as used herein, includesmodifications which introduce chemistries which differ from those seenin naturally occurring mRNA, for example, covalent modifications such asthe introduction of modified nucleotides, (e.g., nucleotide analogs, orthe inclusion of pendant groups which are not naturally found in suchmRNA molecules).

In addition, suitable modifications include alterations in one or morenucleotides of a codon such that the codon encodes the same amino acidbut is more stable than the codon found in the wild-type version of themRNA. For example, an inverse relationship between the stability of RNAand a higher number cytidines (C's) and/or uridines (U's) residues hasbeen demonstrated, and RNA devoid of C and U residues have been found tobe stable to most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8(1994)). In some embodiments, the number of C and/or U residues in anmRNA sequence is reduced. In a another embodiment, the number of Cand/or U residues is reduced by substitution of one codon encoding aparticular amino acid for another codon encoding the same or a relatedamino acid. Contemplated modifications to the mRNA nucleic acids of thepresent invention also include the incorporatation of pseudouridines.The incorporation of pseudouridines into the mRNA nucleic acids of thepresent invention may enhance stability and translational capacity, aswell as diminishing immunogenicity in vivo. See, e.g., Kariko, K., etal., Molecular Therapy 16 (11): 1833-1840 (2008). Substitutions andmodifications to the mRNA of the present invention may be performed bymethods readily known to one or ordinary skill in the art.

The constraints on reducing the number of C and U residues in a sequencewill likely be greater within the coding region of an mRNA, compared toan untranslated region, (i.e., it will likely not be possible toeliminate all of the C and U residues present in the message while stillretaining the ability of the message to encode the desired amino acidsequence). The degeneracy of the genetic code, however presents anopportunity to allow the number of C and/or U residues that are presentin the sequence to be reduced, while maintaining the same codingcapacity (i.e., depending on which amino acid is encoded by a codon,several different possibilities for modification of RNA sequences may bepossible). For example, the codons for Gly can be altered to GGA or GGGinstead of GGU or GGC.

The term modification also includes, for example, the incorporation ofnon-nucleotide linkages or modified nucleotides into the mRNA sequencesof the present invention (e.g., modifications to one or both the 3′ and5′ ends of an mRNA molecule encoding a functional secreted protein orenzyme). Such modifications include the addition of bases to an mRNAsequence (e.g., the inclusion of a poly A tail or a longer poly A tail),the alteration of the 3′ UTR or the 5′ UTR, complexing the mRNA with anagent (e.g., a protein or a complementary nucleic acid molecule), andinclusion of elements which change the structure of an mRNA molecule(e.g., which form secondary structures).

The poly A tail is thought to stabilize natural messengers. Therefore,in one embodiment a long poly A tail can be added to an mRNA moleculethus rendering the mRNA more stable. Poly A tails can be added using avariety of art-recognized techniques. For example, long poly A tails canbe added to synthetic or in vitro transcribed mRNA using poly Apolymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). Atranscription vector can also encode long poly A tails. In addition,poly A tails can be added by transcription directly from PCR products.In one embodiment, the length of the poly A tail is at least about 90,200, 300, 400 at least 500 nucleotides. In one embodiment, the length ofthe poly A tail is adjusted to control the stability of a modified mRNAmolecule of the invention and, thus, the transcription of protein. Forexample, since the length of the poly A tail can influence the half-lifeof an mRNA molecule, the length of the poly A tail can be adjusted tomodify the level of resistance of the mRNA to nucleases and therebycontrol the time course of protein expression in a cell. In oneembodiment, the stabilized mRNA molecules are sufficiently resistant toin vivo degradation (e.g., by nucleases), such that they may bedelivered to the target cell without a transfer vehicle.

In one embodiment, an mRNA can be modified by the incorporation 3′and/or 5′ untranslated (UTR) sequences which are not naturally found inthe wild-type mRNA. In one embodiment, 3′ and/or 5′ flanking sequencewhich naturally flanks an mRNA and encodes a second, unrelated proteincan be incorporated into the nucleotide sequence of an mRNA moleculeencoding a therapeutic or functional protein in order to modify it. Forexample, 3′ or 5′ sequences from mRNA molecules which are stable (e.g.,globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes)can be incorporated into the 3′ and/or 5′ region of a sense mRNA nucleicacid molecule to increase the stability of the sense mRNA molecule. See,e.g., US2003/0083272.

In some embodiments,the mRNA in the compositions of the inventioninclude modificationof the 5′ end of the mRNA to include a partialsequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof(e.g., SEQ ID NO:1) to improve the nuclease resistance and/or improvethe half-life of the mRNA. In addition to increasing the stability ofthe mRNA nucleic acid sequence, it has been surprisingly discovered theinclusion of a partial sequence of a CMV immediate-early 1 (IE1) geneenhances the translation of the mRNA and the expression of thefunctional protein or enzyme. Also contemplated is the inclusion of ahuman growth hormone (hGH) gene sequence, or a fragment thereof (e.g.,SEQ ID NO:2) to the 3′ ends of the nucleic acid (e.g., mRNA) to furtherstabilize the mRNA. Generally, preferred modifications improve thestability and/or pharmacokinetic properties (e.g., half-life) of themRNA relative to their unmodified counterparts, and include, for examplemodifications made to improve such mRNA's resistance to in vivo nucleasedigestion.

Further contemplated are variants of the nucelic acid sequence of SEQ IDNO:1 and/or SEQ ID NO:2, wherein the the variants maintain thefunctional properties of the nucleic acids including stabilization ofthe mRNA and/or pharmacokinetic properties (e.g., half-life). Variantsmay have greater than 90%, greater than 95%, greater than 98%, orgreater than 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

In some embodiments, the composition can comprise a stabilizing reagent.The compositions can include one or more formulation reagents that binddirectly or indirectly to, and stabilize the mRNA, thereby enhancingresidence time in the target cell. Such reagents preferably lead to animproved half-life of the mRNA in the target cells. For example, thestability of an mRNA and efficiency of translation may be increased bythe incorporation of “stabilizing reagents” that form complexes with themRNA that naturally occur within a cell (see e.g., U.S. Pat. No.5,677,124). Incorporation of a stabilizing reagent can be accomplishedfor example, by combining the poly A and a protein with the mRNA to bestabilized in vitro before loading or encapsulating the mRNA within atransfer vehicle. Exemplary stabilizing reagents include one or moreproteins, peptides, aptamers, translational accessory protein, mRNAbinding proteins, and/or translation initiation factors.

Stabilization of the compositions may also be improved by the use ofopsonization-inhibiting moieties, which are typically large hydrophilicpolymers that are chemically or physically bound to the transfer vehicle(e.g., by the intercalation of a lipid-soluble anchor into the membraneitself, or by binding directly to active groups of membrane lipids).These opsonization-inhibiting hydrophilic polymers form a protectivesurface layer which significantly decreases the uptake of the liposomesby the macrophage-monocyte system and reticulo-endothelial system (e.g.,as described in U.S. Pat. No. 4,920,016, the entire disclosure of whichis herein incorporated by reference). Transfer vehicles modified withopsonization-inhibition moieties thus remain in the circulation muchlonger than their unmodified counterparts.

When RNA is hybridized to a complementary nucleic acid molecule (e.g.,DNA or RNA) it may be protected from nucleases. (Krieg, et al. Melton.Methods in Enzymology. 1987; 155, 397-415). The stability of hybridizedmRNA is likely due to the inherent single strand specificity of mostRNases. In some embodiments, the stabilizing reagent selected to complexa mRNA is a eukaryotic protein, (e.g., a mammalian protein). In yetanother embodiment, the mRNA can be modified by hybridization to asecond nucleic acid molecule. If an entire mRNA molecule were hybridizedto a complementary nucleic acid molecule translation initiation may bereduced. In some embodiments the 5′ untranslated region and the AUGstart region of the mRNA molecule may optionally be left unhybridized.Following translation initiation, the unwinding activity of the ribosomecomplex can function even on high affinity duplexes so that translationcan proceed. (Liebhaber. J. Mol. Biol. 1992; 226: 2-13; Monia, et al. JBiol Chem. 1993; 268: 14514-22.)

It will be understood that any of the above described methods forenhancing the stability of mRNA may be used either alone or incombination with one or more of any of the other above-described methodsand/or compositions.

The mRNA of the present invention may be optionally combined with areporter gene (e.g., upstream or downstream of the coding region of themRNA) which, for example, facilitates the determination of mRNA deliveryto the target cells or tissues. Suitable reporter genes may include, forexample, Green Fluorescent Protein mRNA (GFP mRNA), Renilla LuciferasemRNA (Luciferase mRNA), Firefly Luciferase mRNA, or any combinationsthereof. For example, GFP mRNA may be fused with a mRNA encoding asecretable protein to facilitate confirmation of mRNA localization inthe target cells that will act as a depot for protein production.

As used herein, the terms “transfect” or “transfection” mean theintracellular introduction of a mRNA into a cell, or preferably into atarget cell. The introduced mRNA may be stably or transiently maintainedin the target cell. The term “transfection efficiency” refers to therelative amount of mRNA taken up by the target cell which is subject totransfection. In practice, transfection efficiency is estimated by theamount of a reporter nucleic acid product expressed by the target cellsfollowing transfection. Preferred embodiments include compositions withhigh transfection efficacies and in particular those compositions thatminimize adverse effects which are mediated by transfection ofnon-target cells. The compositions of the present invention thatdemonstrate high transfection efficacies improve the likelihood thatappropriate dosages of the mRNA will be delivered to the target cell,while minimizing potential systemic adverse effects. In one embodimentof the present invention, the transfer vehicles of the present inventionare capable of delivering large mRNA sequences (e.g., mRNA of at least 1kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 5 kDa, 10 kDa, 12 kDa, 15 kDa, 20 kDa, 25kDa, 30 kDa, or more). The mRNA can be formulated with one or moreacceptable reagents, which provide a vehicle for delivering such mRNA totarget cells. Appropriate reagents are generally selected with regard toa number of factors, which include, among other things, the biologicalor chemical properties of the mRNA, the intended route ofadministration, the anticipated biological environment to which suchmRNA will be exposed and the specific properties of the intended targetcells. In some embodiments, transfer vehicles, such as liposomes,encapsulate the mRNA without compromising biological activity. In someembodiments, the transfer vehicle demonstrates preferential and/orsubstantial binding to a target cell relative to non-target cells. In apreferred embodiment, the transfer vehicle delivers its contents to thetarget cell such that the mRNA are delivered to the appropriatesubcellular compartment, such as the cytoplasm.

Transfer Vehicle

In embodiments, the transfer vehicle in the compositions of theinvention is a liposomal transfer vehicle, e.g. a lipid nanoparticle. Inone embodiment, the transfer vehicle may be selected and/or prepared tooptimize delivery of the mRNA to a target cell. For example, if thetarget cell is a hepatocyte the properties of the transfer vehicle(e.g., size, charge and/or pH) may be optimized to effectively deliversuch transfer vehicle to the target cell, reduce immune clearance and/orpromote retention in that target cell. Alternatively, if the target cellis the central nervous system (e.g., mRNA administered for the treatmentof neurodegenerative diseases may specifically target brain or spinaltissue), selection and preparation of the transfer vehicle must considerpenetration of, and retention within the blood brain barrier and/or theuse of alternate means of directly delivering such transfer vehicle tosuch target cell. In one embodiment, the compositions of the presentinvention may be combined with agents that facilitate the transfer ofexogenous mRNA (e.g., agents which disrupt or improve the permeabilityof the blood brain barrier and thereby enhance the transfer of exogenousmRNA to the target cells).

The use of liposomal transfer vehicles to facilitate the delivery ofnucleic acids to target cells is contemplated by the present invention.Liposomes (e.g., liposomal lipid nanoparticles) are generally useful ina variety of applications in research, industry, and medicine,particularly for their use as transfer vehicles of diagnostic ortherapeutic compounds in vivo (Lasic, Trends Biotechnol., 16: 307-321,1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and areusually characterized as microscopic vesicles having an interior aquaspace sequestered from an outer medium by a membrane of one or morebilayers. Bilayer membranes of liposomes are typically formed byamphiphilic molecules, such as lipids of synthetic or natural originthat comprise spatially separated hydrophilic and hydrophobic domains(Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of theliposomes can also be formed by amphiphilic polymers and surfactants(e.g., polymerosomes, niosomes, etc.).

In the context of the present invention, a liposomal transfer vehicletypically serves to transport the mRNA to the target cell. For thepurposes of the present invention, the liposomal transfer vehicles areprepared to contain the desired nucleic acids. The process ofincorporation of a desired entity (e.g., a nucleic acid) into a liposomeis often referred to as “loading” (Lasic, et al., FEBS Lett., 312:255-258, 1992). The liposome-incorporated nucleic acids may becompletely or partially located in the interior space of the liposome,within the bilayer membrane of the liposome, or associated with theexterior surface of the liposome membrane. The incorporation of anucleic acid into liposomes is also referred to herein as“encapsulation” wherein the nucleic acid is entirely contained withinthe interior space of the liposome. The purpose of incorporating a mRNAinto a transfer vehicle, such as a liposome, is often to protect thenucleic acid from an environment which may contain enzymes or chemicalsthat degrade nucleic acids and/or systems or receptors that cause therapid excretion of the nucleic acids. Accordingly, in a preferredembodiment of the present invention, the selected transfer vehicle iscapable of enhancing the stability of the mRNA contained therein. Theliposome can allow the encapsulated mRNA to reach the target cell and/ormay preferentially allow the encapsulated mRNA to reach the target cell,or alternatively limit the delivery of such mRNA to other sites or cellswhere the presence of the administered mRNA may be useless orundesirable. Furthermore, incorporating the mRNA into a transfervehicle, such as for example, a cationic liposome, also facilitates thedelivery of such mRNA into a target cell.

Ideally, liposomal transfer vehicles are prepared to encapsulate one ormore desired mRNA such that the compositions demonstrate a hightransfection efficiency and enhanced stability. While liposomes canfacilitate introduction of nucleic acids into target cells, the additionof polycations (e.g., poly L-lysine and protamine), as a copolymer canfacilitate, and in some instances markedly enhance the transfectionefficiency of several types of cationic liposomes by 2-28 fold in anumber of cell lines both in vitro and in vivo. (See N. J. Caplen, etal., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther. 1997; 4, 891.)

Lipid Nanoparticles

In a preferred embodiment of the present invention, the transfer vehicleis formulated as a lipid nanoparticle. As used herein, the phrase “lipidnanoparticle” refers to a transfer vehicle comprising one or more lipids(e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids).Preferably, the lipid nanoparticles are formulated to deliver one ormore mRNA to one or more target cells. Examples of suitable lipidsinclude, for example, the phosphatidyl compounds (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, sphingolipids, cerebrosides, andgangliosides). Also contemplated is the use of polymers as transfervehicles, whether alone or in combination with other transfer vehicles.Suitable polymers may include, for example, polyacrylates,polyalkycyanoacrylates, polylactide, polylactide-polyglycolidecopolymers, polycaprolactones, dextran, albumin, gelatin, alginate,collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine Inone embodiment, the transfer vehicle is selected based upon its abilityto facilitate the transfection of a mRNA to a target cell.

The invention contemplates the use of lipid nanoparticles as transfervechicles comprising a cationic lipid to encapsulate and/or enhance thedelivery of mRNA into the target cell that will act as a depot forprotein production. As used herein, the phrase “cationic lipid” refersto any of a number of lipid species that carry a net positive charge ata selected pH, such as physiological pH. The contemplated lipidnanoparticles may be prepared by including multi-component lipidmixtures of varying ratios employing one or more cationic lipids,non-cationic lipids and PEG-modified lipids. Several cationic lipidshave been described in the literature, many of which are commerciallyavailable.

Particularly suitable cationic lipids for use in the compositions andmethods of the invention include those described in international patentpublication WO 2010/053572, incorporated herein by reference, and mostparticularly, C12-200 described at paragraph [00225] of WO 2010/053572.In certain embodiments, the compositions and methods of the inventionemploy a lipid nanoparticles comprising an ionizable cationic lipiddescribed in U.S. provisional patent application 61/617,468, filed Mar.29, 2012 (incorporated herein by reference), such as, e.g,(15Z,18Z)-N,N-dimethyl-6-(9Z,12)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine(HGT5000),(15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine(HGT5001), and(15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine(HGT5002).

In some embodiments, the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA”is used. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S.Pat. No. 4,897,355). DOTMA can be formulated alone or can be combinedwith the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” orother cationic or non-cationic lipids into a liposomal transfer vehicleor a lipid nanoparticle, and such liposomes can be used to enhance thedelivery of nucleic acids into target cells. Other suitable cationiclipids include, for example, 5-carboxyspermylglycinedioctadecylamide or“DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.Contemplated cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylammonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”,2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane or “CpLinDMA”,N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-K-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech.28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., JControlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat.Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1).

The use of cholesterol-based cationic lipids is also contemplated by thepresent invention. Such cholesterol-based cationic lipids can be used,either alone or in combination with other cationic or non-cationiclipids. Suitable cholesterol-based cationic lipids include, for example,DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE.

In addition, several reagents are commercially available to enhancetransfection efficacy. Suitable examples include LIPOFECTIN (DOTMA:DOPE)(Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen),LIPOFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), andEFFECTENE.

Also contemplated are cationic lipids such as the dialkylamino-based,imidazole-based, and guanidinium-based lipids. For example, certainembodiments are directed to a composition comprising one or moreimidazole-based cationic lipids, for example, the imidazole cholesterolester or “ICE” lipid(3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (I) below.In a preferred embodiment, a transfer vehicle for delivery of mRNA maycomprise one or more imidazole-based cationic lipids, for example, theimidazole cholesterol ester or “ICE” lipid(3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (I).

Without wishing to be bound by a particular theory, it is believed thatthe fusogenicity of the imidazole-based cationic lipid ICE is related tothe endosomal disruption which is facilitated by the imidazole group,which has a lower pKa relative to traditional cationic lipids. Theendosomal disruption in turn promotes osmotic swelling and thedisruption of the liposomal membrane, followed by the transfection orintracellular release of the nucleic acid(s) contents loaded thereininto the target cell.

The imidazole-based cationic lipids are also characterized by theirreduced toxicity relative to other cationic lipids. The imidazole-basedcationic lipids (e.g., ICE) may be used as the sole cationic lipid inthe lipid nanoparticle, or alternatively may be combined withtraditional cationic lipids, non-cationic lipids, and PEG-modifiedlipids. The cationic lipid may comprise a molar ratio of about 1% toabout 90%, about 2% to about 70%, about 5% to about 50%, about 10% toabout 40% of the total lipid present in the transfer vehicle, orpreferably about 20% to about 70% of the total lipid present in thetransfer vehicle.

Similarly, certain embodiments are directed to lipid nanoparticlescomprising the HGT4003 cationic lipid2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine,as represented by structure (II) below, and as further described in U.S.Provisional Application No. 61/494,745, filed Jun. 8, 2011, the entireteachings of which are incorporated herein by reference in theirentirety:

In other embodiments the compositions and methods described herein aredirected to lipid nanoparticles comprising one or more cleavable lipids,such as, for example, one or more cationic lipids or compounds thatcomprise a cleavable disulfide (S—S) functional group (e.g., HGT4001,HGT4002, HGT4003, HGT4004 and HGT4005), as further described in U.S.Provisional Application No. 61/494,745, the entire teachings of whichare incorporated herein by reference in their entirety.

The use of polyethylene glycol (PEG)-modified phospholipids andderivatized lipids such as derivatized cerarmides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention,either alone or preferably in combination with other lipids togetherwhich comprise the transfer vehicle (e.g., a lipid nanoparticle).Contemplated PEG-modified lipids include, but is not limited to, apolyethylene glycol chain of up to 5 kDa in length covalently attachedto a lipid with alkyl chain(s) of C₆-C₂₀ length. The addition of suchcomponents may prevent complex aggregation and may also provide a meansfor increasing circulation lifetime and increasing the delivery of thelipid-nucleic acid composition to the target cell, (Klibanov et al.(1990) FEBS Letters, 268 (1): 235-237), or they may be selected torapidly exchange out of the formulation in vivo (see U.S. Pat. No.5,885,613). Particularly useful exchangeable lipids are PEG-ceramideshaving shorter acyl chains (e.g., C14 or C18). The PEG-modifiedphospholipid and derivitized lipids of the present invention maycomprise a molar ratio from about 0% to about 20%, about 0.5% to about20%, about 1% to about 15%, about 4% to about 10%, or about 2% of thetotal lipid present in the liposomal transfer vehicle.

The present invention also contemplates the use of non-cationic lipids.As used herein, the phrase “non-cationic lipid” refers to any neutral,zwitterionic or anionic lipid. As used herein, the phrase “anioniclipid” refers to any of a number of lipid species that carry a netnegative charge at a selected pH, such as physiological pH. Non-cationiclipids include, but are not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. Such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. When used in combination with a cationic lipid, thenon-cationic lipid may comprise a molar ratio of 5% to about 90%, orpreferably about 10% to about 70% of the total lipid present in thetransfer vehicle.

Preferably, the transfer vehicle (e.g., a lipid nanoparticle) isprepared by combining multiple lipid and/or polymer components. Forexample, a transfer vehicle may be prepared using C12-200, DOPE, chol,DMG-PEG2K at a molar ratio of 40:30:25:5, or DODAP, DOPE, cholesterol,DMG-PEG2K at a molar ratio of 18:56:20:6, or HGT5000, DOPE, chol,DMG-PEG2K at a molar ratio of 40:20:35:5, or HGT5001, DOPE, chol,DMG-PEG2K at a molar ratio of 40:20:35:5. The selection of cationiclipids, non-cationic lipids and/or PEG-modified lipids which comprisethe lipid nanoparticle, as well as the relative molar ratio of suchlipids to each other, is based upon the characteristics of the selectedlipid(s), the nature of the intended target cells, the characteristicsof the mRNA to be delivered. Additional considerations include, forexample, the saturation of the alkyl chain, as well as the size, charge,pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus themolar ratios may be adjusted accordingly. For example, in embodiments,the percentage of cationic lipid in the lipid nanoparticle may begreater than 10%, greater than 20%, greater than 30%, greater than 40%,greater than 50%, greater than 60%, or greater than 70%. The percentageof non-cationic lipid in the lipid nanoparticle may be greater than 5%,greater than 10%, greater than 20%, greater than 30%, or greater than40%. The percentage of cholesterol in the lipid nanoparticle may begreater than 10%, greater than 20%, greater than 30%, or greater than40%. The percentage of PEG-modified lipid in the lipid nanoparticle maybe greater than 1%, greater than 2%, greater than 5%, greater than 10%,or greater than 20%.

In certain preferred embodiments, the lipid nanoparticles of theinvention comprise at least one of the following cationic lipids:C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. Inembodiments, the transfer vehicle comprises cholesterol and/or aPEG-modified lipid. In some embodiments, the transfer vehicles comprisesDMG-PEG2K. In certain embodiments, the tranfer vehicle comprises one ofthe following lipid formulations: C12-200, DOPE, chol, DMG-PEG2K; DODAP,DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, chol, DMG-PEG2K, HGT5001,DOPE, chol, DMG-PEG2K.

The liposomal transfer vehicles for use in the compositions of theinvention can be prepared by various techniques which are presentlyknown in the art. Multi-lamellar vesicles (MLV) may be preparedconventional techniques, for example, by depositing a selected lipid onthe inside wall of a suitable container or vessel by dissolving thelipid in an appropriate solvent, and then evaporating the solvent toleave a thin film on the inside of the vessel or by spray drying. Anaqueous phase may then added to the vessel with a vortexing motion whichresults in the formation of MLVs. Uni-lamellar vesicles (ULV) can thenbe formed by homogenization, sonication or extrusion of themulti-lamellar vesicles. In addition, unilamellar vesicles can be formedby detergent removal techniques.

In certain embodiments of this invention, the compositions of thepresent invention comprise a transfer vehicle wherein the mRNA isassociated on both the surface of the transfer vehicle and encapsulatedwithin the same transfer vehicle. For example, during preparation of thecompositions of the present invention, cationic liposomal transfervehicles may associate with the mRNA through electrostatic interactions.

In certain embodiments, the compositions of the invention may be loadedwith diagnostic radionuclide, fluorescent materials or other materialsthat are detectable in both in vitro and in vivo applications. Forexample, suitable diagnostic materials for use in the present inventionmay include Rhodamine-dioleoylphospha-tidylethanolamine (Rh-PE), GreenFluorescent Protein mRNA (GFP mRNA), Renilla Luciferase mRNA and FireflyLuciferase mRNA.

Selection of the appropriate size of a liposomal transfer vehicle musttake into consideration the site of the target cell or tissue and tosome extent the application for which the liposome is being made. Insome embodiments, it may be desirable to limit transfection of the mRNAto certain cells or tissues. For example, to target hepatocytes aliposomal transfer vehicle may be sized such that its dimensions aresmaller than the fenestrations of the endothelial layer lining hepaticsinusoids in the liver; accordingly the liposomal transfer vehicle canreadily penetrate such endothelial fenestrations to reach the targethepatocytes. Alternatively, a liposomal transfer vehicle may be sizedsuch that the dimensions of the liposome are of a sufficient diameter tolimit or expressly avoid distribution into certain cells or tissues. Forexample, a liposomal transfer vehicle may be sized such that itsdimensions are larger than the fenestrations of the endothelial layerlining hepatic sinusoids to thereby limit distribution of the liposomaltransfer vehicle to hepatocytes. Generally, the size of the transfervehicle is within the range of about 25 to 250 nm, prefereably less thanabout 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or 10nm.

A variety of alternative methods known in the art are available forsizing of a population of liposomal transfer vehicles. One such sizingmethod is described in U.S. Pat. No. 4,737,323, incorporated herein byreference. Sonicating a liposome suspension either by bath or probesonication produces a progressive size reduction down to small ULV lessthan about 0.05 microns in diameter. Homogenization is another methodthat relies on shearing energy to fragment large liposomes into smallerones. In a typical homogenization procedure, MLV are recirculatedthrough a standard emulsion homogenizer until selected liposome sizes,typically between about 0.1 and 0.5 microns, are observed. The size ofthe liposomal vesicles may be determined by quasi-electric lightscattering (QELS) as described in Bloomfield, Ann. Rev. Biophys.Bioeng., 10:421-450 (1981), incorporated herein by reference. Averageliposome diameter may be reduced by sonication of formed liposomes.Intermittent sonication cycles may be alternated with QELS assessment toguide efficient liposome synthesis.

Target Cells

As used herein, the term “target cell” refers to a cell or tissue towhich a composition of the invention is to be directed or targeted. Insome embodiments, the target cells are deficient in a protein or enzymeof interest. For example, where it is desired to deliver a nucleic acidto a hepatocyte, the hepatocyte represents the target cell. In someembodiments, the compositions of the invention transfect the targetcells on a discriminatory basis (i.e., do not transfect non-targetcells). The compositions of the invention may also be prepared topreferentially target a variety of target cells, which include, but arenot limited to, hepatocytes, epithelial cells, hematopoietic cells,epithelial cells, endothelial cells, lung cells, bone cells, stem cells,mesenchymal cells, neural cells (e.g., meninges, astrocytes, motorneurons, cells of the dorsal root ganglia and anterior horn motorneurons), photoreceptor cells (e.g., rods and cones), retinal pigmentedepithelial cells, secretory cells, cardiac cells, adipocytes, vascularsmooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells,pituitary cells, synovial lining cells, ovarian cells, testicular cells,fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytesand tumor cells.

The compositions of the invention may be prepared to preferentiallydistribute to target cells such as in the heart, lungs, kidneys, liver,and spleen. In some embodiments, the compositions of the inventiondistribute into the cells of the liver to facilitate the delivery andthe subsequent expression of the mRNA comprised therein by the cells ofthe liver (e.g., hepatocytes). The targeted hepatocytes may function asa biological “reservoir” or “depot” capable of producing, andsystemically excreting a functional protein or enzyme. Accordingly, inone embodiment of the invention the liposomal transfer vehicle maytarget hepatocyes and/or preferentially distribute to the cells of theliver upon delivery. Following transfection of the target hepatocytes,the mRNA loaded in the liposomal vehicle are translated and a functionalprotein product is produced, excreted and systemically distributed. Inother emodiments, cells other than hepatocytes (e.g., lung, spleen,heart, ocular, or cells of the central nervous system) can serve as adepot location for protein production.

In one embodiment, the compositions of the invention facilitate asubject's endogenous production of one or more functional proteinsand/or enzymes, and in particular the production of proteins and/orenzymes which demonstrate less immunogenicity relative to theirrecombinantly-prepared counterparts. In a preferred embodiment of thepresent invention, the transfer vehicles comprise mRNA which encode adeficient protein or enzyme. Upon distribution of such compositions tothe target tissues and the subsequent transfection of such target cells,the exogenous mRNA loaded into the liposomal transfer vehicle (e.g., alipid nanoparticle) may be translated in vivo to produce a functionalprotein or enzyme encoded by the exogenously administered mRNA (e.g., aprotein or enzyme in which the subject is deficient). Accordingly, thecompositions of the present invention exploit a subject's ability totranslate exogenously- or recombinantly-prepared mRNA to produce anendogenously-translated protein or enzyme, and thereby produce (andwhere applicable excrete) a functional protein or enzyme. The expressedor translated proteins or enzymes may also be characterized by the invivo inclusion of native post-translational modifications which mayoften be absent in recombinantly-prepared proteins or enzymes, therebyfurther reducing the immunogenicity of the translated protein or enzyme.

The administration of mRNA encoding a deficient protein or enzyme avoidsthe need to deliver the nucleic acids to specific organelles within atarget cell (e.g., mitochondria). Rather, upon transfection of a targetcell and delivery of the nucleic acids to the cytoplasm of the targetcell, the mRNA contents of a transfer vehicle may be translated and afunctional protein or enzyme expressed.

The present invention also contemplates the discriminatory targeting oftarget cells and tissues by both passive and active targeting means. Thephenomenon of passive targeting exploits the natural distributionspatterns of a transfer vehicle in vivo without relying upon the use ofadditional excipients or means to enhance recognition of the transfervehicle by target cells. For example, transfer vehicles which aresubject to phagocytosis by the cells of the reticulo-endothelial systemare likely to accumulate in the liver or spleen, and accordingly mayprovide means to passively direct the delivery of the compositions tosuch target cells.

Alternatively, the present invention contemplates active targeting,which involves the use of additional excipients, referred to herein as“targeting ligands” that may be bound (either covalently ornon-covalently) to the transfer vehicle to encourage localization ofsuch transfer vehicle at certain target cells or target tissues. Forexample, targeting may be mediated by the inclusion of one or moreendogenous targeting ligands (e.g., apolipoprotein E) in or on thetransfer vehicle to encourage distribution to the target cells ortissues. Recognition of the targeting ligand by the target tissuesactively facilitates tissue distribution and cellular uptake of thetransfer vehicle and/or its contents in the target cells and tissues(e.g., the inclusion of an apolipoprotein-E targeting ligand in or onthe transfer vehicle encourages recognition and binding of the transfervehicle to endogenous low density lipoprotein receptors expressed byhepatocytes). As provided herein, the composition can comprise a ligandcapable of enhancing affinity of the composition to the target cell.Targeting ligands may be linked to the outer bilayer of the lipidparticle during formulation or post-formulation. These methods are wellknown in the art. In addition, some lipid particle formulations mayemploy fusogenic polymers such as PEAA, hemagluttinin, otherlipopeptides (see U.S. patent application Ser. No. 08/835,281, and60/083,294, which are incorporated herein by reference) and otherfeatures useful for in vivo and/or intracellular delivery. In other someembodiments, the compositions of the present invention demonstrateimproved transfection efficacies, and/or demonstrate enhancedselectivity towards target cells or tissues of interest. Contemplatedtherefore are compositions which comprise one or more ligands (e.g.,peptides, aptamers, oligonucleotides, a vitamin or other molecules) thatare capable of enhancing the affinity of the compositions and theirnucleic acid contents for the target cells or tissues. Suitable ligandsmay optionally be bound or linked to the surface of the transfervehicle. In some embodiments, the targeting ligand may span the surfaceof a transfer vehicle or be encapsulated within the transfer vehicle.Suitable ligands and are selected based upon their physical, chemical orbiological properties (e.g., selective affinity and/or recognition oftarget cell surface markers or features.) Cell-specific target sites andtheir corresponding targeting ligand can vary widely. Suitable targetingligands are selected such that the unique characteristics of a targetcell are exploited, thus allowing the composition to discriminatebetween target and non-target cells. For example, compositions of theinvention may include surface markers (e.g., apolipoprotein-B orapolipoprotein-E) that selectively enhance recognition of, or affinityto hepatocytes (e.g., by receptor-mediated recognition of and binding tosuch surface markers). Additionally, the use of galactose as a targetingligand would be expected to direct the compositions of the presentinvention to parenchymal hepatocytes, or alternatively the use ofmannose containing sugar residues as a targeting ligand would beexpected to direct the compositions of the present invention to liverendothelial cells (e.g., mannose containing sugar residues that may bindpreferentially to the asialoglycoprotein receptor present inhepatocytes). (See Hillery A M, et al. “Drug Delivery and Targeting: ForPharmacists and Pharmaceutical Scientists” (2002) Taylor & Francis,Inc.) The presentation of such targeting ligands that have beenconjugated to moieties present in the transfer vehicle (e.g., a lipidnanoparticle) therefore facilitate recognition and uptake of thecompositions of the present invention in target cells and tissues.Examples of suitable targeting ligands include one or more peptides,proteins, aptamers, vitamins and oligonucleotides.

Application and Administration

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, to which the compositions and methods of thepresent invention are administered. Typically, the terms “subject” and“patient” are used interchangeably herein in reference to a humansubject.

The compositions and methods of the invention provide for the deliveryof mRNA to treat a number of disorders. In particular, the compositionsand methods of the present invention are suitable for the treatment ofdiseases or disorders relating to the deficiency of proteins and/orenzymes that are excreted or secreted by the target cell into thesurrounding extracellular fluid (e.g., mRNA encoding hormones andneurotransmitters). In embodiments the disease may involve a defect ordeficiency in a secreted protein (e.g. Fabry disease, or ALS). Incertain embodiments, the disease may not be caused by a defect ordeficit in a secreted protein, but may benefit from providing a secretedprotein. For example, the symptoms of a disease may be improved byproviding the compositions of the invention (e.g. cystic fibrosis).Disorders for which the present invention are useful include, but arenot limited to, disorders such as Huntington's Disease; Parkinson'sDisease; muscular dystrophies (such as, e.g. Duchenne and Becker);hemophelia diseases (such as, e.g., hemophilioa B (FIX), hemophilia A(FVIII); SMN1-related spinal muscular atrophy (SMA); amyotrophic lateralsclerosis (ALS); GALT-related galactosemia; Cystic Fibrosis (CF);SLC3A1-related disorders including cystinuria; COL4A5-related disordersincluding Alport syndrome; galactocerebrosidase deficiencies; X-linkedadrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia;Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis;Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; theFMR1-related disorders which include Fragile X syndrome, FragileX-Associated Tremor/Ataxia Syndrome and Fragile X Premature OvarianFailure Syndrome; Prader-Willi syndrome; hereditary hemorrhagictelangiectasia (AT); Niemann-Pick disease Type C1; the neuronal ceroidlipofuscinoses-related diseases including Juvenile Neuronal CeroidLipofuscinosis (JNCL), Juvenile Batten disease, Santavuori-Haltiadisease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiencies;EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5-related childhood ataxia withcentral nervous system hypomyelination/vanishing white matter; CACNA1Aand CACNB4-related Episodic Ataxia Type 2; the MECP2-related disordersincluding Classic Rett Syndrome, MECP2-related Severe NeonatalEncephalopathy and PPM-X Syndrome; CDKL5-related Atypical Rett Syndrome;Kennedy's disease (SBMA); Notch-3 related cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL); SCN1A and SCN1B-related seizure disorders; the PolymeraseG-related disorders which include Alpers-Huttenlocher syndrome,POLG-related sensory ataxic neuropathy, dysarthria, andophthalmoparesis, and autosomal dominant and recessive progressiveexternal ophthalmoplegia with mitochondrial DNA deletions; X-Linkedadrenal hypoplasia; X-linked agammaglobulinemia; Wilson's disease; andFabry Disease. In one embodiment, the nucleic acids, and in particularmRNA, of the invention may encode functional proteins or enzymes thatare secreted into extracellular space. For example, the secretedproteins include clotting factors, components of the complement pathway,cytokines, chemokines, chemoattractants, protein hormones (e.g. EGF,PDF), protein components of serum, antibodies, secretable toll-likereceptors, and others. In some embodiments, the compositions of thepresent invention may include mRNA encoding erythropoietinα1-antitrypsin, carboxypeptidase N or human growth hormone.

In embodiments, the invention encodes a secreted protein that is made upof subunits that are encoded by more than one gene. For example, thesecreted protein may be a heterodimer, wherein each chain or subunit ofthe is encoded by a separate gene. It is possible that more than onemRNA molecule is delivered in the transfer vehicle and the mRNA encodesseparate subunit of the secreted protein. Alternatively, a single mRNAmay be engineered to encode more than one subunit (e.g. in the case of asingle-chain Fv antibody). In certain embodiments, separate mRNAmolecules encoding the individual subunits may be administered inseparate transfer vehicles. In one embodiment, the mRNA may encode fulllength antibodies (both heavy and light chains of the variable andconstant regions) or fragments of antibodies (e.g. Fab, Fv, or a singlechain Fv (scFv) to confer immunity to a subject. While one embodiment ofthe present invention relates to methods and compositions useful forconferring immunity to a subject (e.g., via the translation of mRNAencoding functional antibodies), the inventions disclosed herein andcontemplated hereby are broadly applicable. In an alternative embodimentthe compositions of the present invention encode antibodies that may beused to transiently or chronically effect a functional response insubjects. For example, the mRNA of the present invention may encode afunctional monoclonal or polyclonal antibody, which upon translation andsecretion from target cell may be useful for targeting and/orinactivating a biological target (e.g., a stimulatory cytokine such astumor necrosis factor). Similarly, the mRNA nucleic acids of the presentinvention may encode, for example, functional anti-nephritic factorantibodies useful for the treatment of membranoproliferativeglomerulonephritis type II or acute hemolytic uremic syndrome, oralternatively may encode anti-vascular endothelial growth factor (VEGF)antibodies useful for the treatment of VEGF-mediated diseases, such ascancer. In other embodiments, the secreted protein is a cytokine orother secreted protein comprised of more than one subunit (e.g. IL-12,or IL-23).

The compositions of the invention can be administered to a subject. Insome embodiments, the composition is formulated in combination with oneor more additional nucleic acids, carriers, targeting ligands orstabilizing reagents, or in pharmacological compositions where it ismixed with suitable excipients. For example, in one embodiment, thecompositions of the invention may be prepared to deliver mRNA encodingtwo or more distinct proteins or enzymes. Techniques for formulation andadministration of drugs may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

A wide range of molecules that can exert pharmaceutical or therapeuticeffects can be delivered into target cells using compositions andmethods of the invention. The molecules can be organic or inorganic.Organic molecules can be peptides, proteins, carbohydrates, lipids,sterols, nucleic acids (including peptide nucleic acids), or anycombination thereof. A formulation for delivery into target cells cancomprise more than one type of molecule, for example, two differentnucleotide sequences, or a protein, an enzyme or a steroid.

The compositions of the present invention may be administered and dosedin accordance with current medical practice, taking into account theclinical condition of the subject, the site and method ofadministration, the scheduling of administration, the subject's age,sex, body weight and other factors relevant to clinicians of ordinaryskill in the art. The “effective amount” for the purposes herein may bedetermined by such relevant considerations as are known to those ofordinary skill in experimental clinical research, pharmacological,clinical and medical arts. In some embodiments, the amount administeredis effective to achieve at least some stabilization, improvement orelimination of symptoms and other indicators as are selected asappropriate measures of disease progress, regression or improvement bythose of skill in the art. For example, a suitable amount and dosingregimen is one that causes at least transient protein production.

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternately, the compositions of the invention may be administered in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a targeted tissue, preferablyin a sustained release formulation. Local delivery can be affected invarious ways, depending on the tissue to be targeted. For example,aerosols containing compositions of the present invention can be inhaled(for nasal, tracheal, or bronchial delivery); compositions of thepresent invention can be injected into the site of injury, diseasemanifestation, or pain, for example; compositions can be provided inlozenges for oral, tracheal, or esophageal application; can be suppliedin liquid, tablet or capsule form for administration to the stomach orintestines, can be supplied in suppository form for rectal or vaginalapplication; or can even be delivered to the eye by use of creams,drops, or even injection. Formulations containing compositions of thepresent invention complexed with therapeutic molecules or ligands caneven be surgically administered, for example in association with apolymer or other structure or substance that can allow the compositionsto diffuse from the site of implantation to surrounding cells.Alternatively, they can be applied surgically without the use ofpolymers or supports.

In one embodiment, the compositions of the invention are formulated suchthat they are suitable for extended-release of the mRNA containedtherein. Such extended-release compositions may be convenientlyadministered to a subject at extended dosing intervals. For example, inone embodiment, the compositions of the present invention areadministered to a subject twice day, daily or every other day. In apreferred embodiment, the compositions of the present invention areadministered to a subject twice a week, once a week, every ten days,every two weeks, every three weeks, or more preferably every four weeks,once a month, every six weeks, every eight weeks, every other month,every three months, every four months, every six months, every eightmonths, every nine months or annually. Also contemplated arecompositions and liposomal vehicles which are formulated for depotadministration (e.g., intramuscularly, subcutaneously, intravitreally)to either deliver or release a mRNA over extended periods of time.Preferably, the extended-release means employed are combined withmodifications made to the mRNA to enhance stability.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the liposomal nanoparticles disclosed hereinand related methods for the use of such lyophilized compositions asdisclosed for example, in U.S. Provisional Application No. 61/494,882,filed Jun. 8, 2011, the teachings of which are incorporated herein byreference in their entirety. For example, lyophilized pharmaceuticalcompositions according to the invention may be reconstituted prior toadministration or can be reconstituted in vivo. For example, alyophilized pharmaceutical composition can be formulated in anappropriate dosage form (e.g., an intradermal dosage form such as adisk, rod or membrane) and administered such that the dosage form isrehydrated over time in vivo by the individual's bodily fluids.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the publications, reference materials, accession numbers and the likereferenced herein to describe the background of the invention and toprovide additional detail regarding its practice are hereby incorporatedby reference in their entirety.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

EXAMPLES Example 1: Protein Production Depot Via Intravenous Delivery ofPolynucleotide Compositions Messenger RNA

Human erythropoietin (EPO) (SEQ ID NO: 3; FIG. 3), humanalpha-galactosidase (GLA) (SEQ ID NO: 4; FIG. 4), human alpha-1antitrypsin (A1AT) (SEQ ID NO: 5; FIG. 5), and human factor IX (FIX)(SEQ ID NO: 6; FIG. 6) were synthesized by in vitro transcription from aplasmid DNA template encoding the gene, which was followed by theaddition of a 5′ cap structure (Cap1) (Fechter & Brownlee, J. Gen.Virology 86:1239-1249 (2005)) and a 3′ poly(A) tail of approximately 200nucleotides in length as determined by gel electrophoresis. 5′ and 3′untranslated regions were present in each mRNA product in the followingexamples and are defined by SEQ ID NOs: 1 and 2 (FIG. 1 and FIG. 2)respectively.

Lipid Nanoparticle Formulations

Formulation 1: Aliquots of 50 mg/mL ethanolic solutions of C12-200,DOPE, Chol and DMG-PEG2K (40:30:25:5) were mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of mRNA was prepared from a 1 mg/mLstock. The lipid solution was injected rapidly into the aqueous mRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C.Formulation 2: Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE,cholesterol and DMG-PEG2K (18:56:20:6) were mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a 1mg/mL stock. The lipid solution was injected rapidly into the aqueousmRNA solution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.35mg/mL EPO mRNA (encapsulated). Z_(ave)=75.9 nm (Dv₍₅₀₎=57.3 nm;Dv₍₉₀₎=92.1 nm).Formulation 3: Aliquots of 50 mg/mL ethanolic solutions of HGT4003,DOPE, cholesterol and DMG-PEG2K (50:25:20:5) were mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of mRNA was prepared from a 1 mg/mLstock. The lipid solution was injected rapidly into the aqueous mRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C.Formulation 4: Aliquots of 50 mg/mL ethanolic solutions of ICE, DOPE andDMG-PEG2K (70:25:5) were mixed and diluted with ethanol to 3 mL finalvolume. Separately, an aqueous buffered solution (10 mM citrate/150 mMNaCl, pH 4.5) of mRNA was prepared from a 1 mg/mL stock. The lipidsolution was injected rapidly into the aqueous mRNA solution and shakento yield a final suspension in 20% ethanol. The resulting nanoparticlesuspension was filtered, diafiltrated with 1×PBS (pH 7.4), concentratedand stored at 2-8° C.Formulation 5: Aliquots of 50 mg/mL ethanolic solutions of HGT5000,DOPE, cholesterol and DMG-PEG2K (40:20:35:5) were mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a 1mg/mL stock. The lipid solution was injected rapidly into the aqueousmRNA solution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.82mg/mL EPO mRNA (encapsulated). Z_(ave)=105.6 nm (Dv₍₅₀₎=53.7 nm;Dv₍₉₀₎=157 nm).Formulation 6: Aliquots of 50 mg/mL ethanolic solutions of HGT5001,DOPE, cholesterol and DMG-PEG2K (40:20:35:5) were mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a 1mg/mL stock. The lipid solution was injected rapidly into the aqueousmRNA solution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C.Analysis of Protein Produced Via Intravenously Delivered mRNA-LoadedNanoparticles Injection Protocol

Studies were performed using male CD-1 mice of approximately 6-8 weeksof age at the beginning of each experiment, unless otherwise indicated.Samples were introduced by a single bolus tail-vein injection of anequivalent total dose of 30-200 micrograms of encapsulated mRNA. Micewere sacrificed and perfused with saline at the designated time points.

Isolation of Organ Tissues for Analysis

The liver and spleen of each mouse was harvested, apportioned into threeparts, and stored in either 10% neutral buffered formalin or snap-frozenand stored at −80° C. for analysis.

Isolation of Serum for Analysis

All animals were euthanized by CO₂ asphyxiation 48 hours post doseadministration (±5%) followed by thoracotomy and terminal cardiac bloodcollection. Whole blood (maximal obtainable volume) was collected viacardiac puncture on euthanized animals into serum separator tubes,allowed to clot at room temperature for at least 30 minutes, centrifugedat 22° C.±5° C. at 9300 g for 10 minutes, and the serum extracted. Forinterim blood collections, approximately 40-50 μL of whole blood wascollected via facial vein puncture or tail snip. Samples collected fromnon treatment animals were used as a baseline for comparison to studyanimals.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis

EPO ELISA: Quantification of EPO protein was performed followingprocedures reported for human EPO ELISA kit (Quantikine IVD, R&DSystems, Catalog #Dep-00). Positive controls employed consisted ofultrapure and tissue culture grade recombinant human erythropoietinprotein (R&D Systems, Catalog #286-EP and 287-TC, respectively).Detection was monitored via absorption (450 nm) on a Molecular DeviceFlex Station instrument.GLA ELISA: Standard ELISA procedures were followed employing sheepanti-Alpha-galactosidase G-188 IgG as the capture antibody with rabbitanti-Alpha-galactosidase TK-88 IgG as the secondary (detection) antibody(Shire Human Genetic Therapies). Horseradish peroxidase (HRP)-conjugatedgoat anti-rabbit IgG was used for activation of the3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The reactionwas quenched using 2N H₂SO₄ after 20 minutes. Detection was monitoredvia absorption (450 nm) on a Molecular Device Flex Station instrument.Untreated mouse serum and human Alpha-galactosidase protein were used asnegative and positive controls, respectively.FIX ELISA: Quantification of FIX protein was performed followingprocedures reported for human FIX ELISA kit (AssayMax, Assay Pro,Catalog #EF1009-1).A1AT ELISA: Quantification of A1AT protein was performed followingprocedures reported for human A1AT ELISA kit (Innovative Research,Catalog #IRAPKT015).

Western Blot Analysis

(EPO): Western blot analyses were performed using an anti-hEPO antibody(R&D Systems #MAB2871) and ultrapure human EPO protein (R&D Systems#286-EP) as the control.

Results

The work described in this example demonstrates the use ofmRNA-encapsulated lipid nanoparticles as a depot source for theproduction of protein. Such a depot effect can be achieved in multiplesites within the body (i.e., liver, kidney, spleen, and muscle).Measurement of the desired exogenous-based protein derived frommessenger RNA delivered via liposomal nanoparticles was achieved andquantified, and the secretion of protein from a depot using humanerythropoietin (hEPO), human alpha-galactosidase (hGLA), human alpha-1antitrypsin (hA1AT), and human Factor IX (hFIX) mRNA was demonstrated.

1A. In Vivo Human EPO Protein Production Results

The production of hEPO protein was demonstrated with various lipidnanoparticle formulations. Of four different cationic lipid systems,C12-200-based lipid nanoparticles produced the highest quantity of hEPOprotein after four hours post intravenous administration as measured byELISA (FIG. 7). This formulation (Formulation 1) resulted in 18.3 ug/mLhEPO protein secreted into the bloodstream. Normal hEPO protein levelsin serum for human are 3.3-16.6 mIU/mL (NCCLS Document C28-P; Vol. 12,No. 2). Based on a specific activity of 120,000 IU/mg of EPO protein,that yields a quantity of 27.5-138 pg/mL hEPO protein in normal humanindividuals. Therefore, a single 30 ug dose of a C12-200-based cationiclipid formulation encapsulating hEPO mRNA yielded an increase inrespective protein of over 100,000-fold physiological levels.

Of the lipid systems tested, the DODAP-based lipid nanoparticleformulation was the least effective. However, the observed quantity ofhuman EPO protein derived from delivery via a DODAP-based lipidnanoparticle encapsulating EPO mRNA was 4.1 ng/mL, which is stillgreater than 30-fold over normal physiological levels of EPO protein(Table 1).

TABLE 1 Raw values of secreted hEPO protein for various cationic lipid-based nanoparticle systems as measured via ELISA analysis (as depictedin FIG. 8). Doses are based on encapsulated hEPO mRNA. Values of proteinare depicted as nanogram of human EPO protein per milliliter of serum.Hematocrit changes are based on comparison of pre-bleed (Day −1) and Day10. Dose of Secreted Encapsulated Human EPO Increase inCationic/Ionizable mRNA Protein Hematocrit Lipid Component (ug) (ng/mL)(%) C12-200 30 18,306 15.0 HGT4003 150 164 0.0 ICE 100 56.2 0.0 DODAP200 4.1 0.0

In addition, the resulting protein was tested to determine if it wasactive and functioned properly. In the case of mRNA replacement therapy(MRT) employing hEPO mRNA, hematocrit changes were monitored over a tenday period for five different lipid nanoparticle formulations (FIG. 8,Table 1) to evaluate protein activity. During this time period, two ofthe five formulations demonstrated an increase in hematocrit (≥15%),which is indicative of active hEPO protein being produced from suchsystems.

In another experiment, hematocrit changes were monitored over a 15-dayperiod (FIG. 9, Table 2). The lipid nanoparticle formulation(Formulation 1) was administered either as a single 30 μg dose, or asthree smaller 10 μg doses injected on day 1, day 3 and day 5. Similarly,Formulation 2 was administered as 3 doses of 50 μg on day 1, day 3, andday 5. C12-200 produced a significant increase in hematocrit. Overall anincrease of up to ˜25% change was observed, which is indicative ofactive human EPO protein being produced from such systems.

TABLE 2 Hematocrit levels of each group over a 15 day observation period(FIG. 9). Mice were either dosed as a single injection, or threeinjections, every other day. N = 4 mice per group. Test Dose Hct LevelsMean (%) ± SEM Article (μg/animal) Day −4 Day 7 Day 10 Day 15^(a)C12-200 30 (single 50.8 ± 1.8 58.3 ± 3.3 62.8 ± 1.3 59.9 ± 3.3 dose)C12-200 30 (over 3 52.2 ± 0.5 55.3 ± 2.3 63.3 ± 1.6 62.3 ± 1.9 doses)DODAP 150 (over 3 54.8 ± 1.7 53.5 ± 1.6 54.2 ± 3.3 54.0 ± 0.3 doses) Hct= hematocrit; SEM = standard error of the mean. ^(a)Blood samples werecollected into non-heparinized hematocrit tubes.

1B. In Vivo Human GLA Protein Production Results

A second exogenous-based protein system was explored to demonstrate the“depot effect” when employing mRNA-loaded lipid nanoparticles. Animalswere injected intravenously with a single 30 microgram dose ofencapsulated human alpha-galactosidase (hGLA) mRNA using a C12-200-basedlipid nanoparticle system and sacrificed after six hours (Formulation1). Quantification of secreted hGLA protein was performed via ELISA.Untreated mouse serum and human Alpha-galactosidase protein were used ascontrols. Detection of human alpha-galactosidase protein was monitoredover a 48 hour period.

Measurable levels of hGLA protein were observed throughout the timecourse of the experiment with a maximum level of 2.0 ug/mL hGLA proteinat six hours (FIG. 10). Table 3 lists the specific quantities of hGLAfound in the serum. Normal activity in healthy human males has beenreported to be approximately 3.05 nanomol/hr/mL. The activity forAlpha-galactosidase, a recombinant human alpha-galactosidase protein,3.56×10⁶ nanomol/hr/mg. Analysis of these values yields a quantity ofapproximately 856 pg/mL of hGLA protein in normal healthy maleindividuals. The quantity of 2.0 ug/mL hGLA protein observed after sixhours when dosing a hGLA mRNA-loaded lipid nanoparticle is over2300-fold greater than normal physiological levels. Further, after 48hours, one can still detect appreciable levels of hGLA protein (86.2ng/mL). This level is representative of almost 100-fold greaterquantities of hGLA protein over physiological amounts still present at48 hours.

TABLE 3 Raw values of secreted hGLA protein over time as measured viaELISA analysis (as depicted in FIG. 10). Values are depicted as nanogramof hGLA protein per milliliter of serum. N = 4 mice per group. TimeSecreted Human Post-Administration (hr) GLA Protein (ng/mL) 6 2,038 121,815 24 414 48 86.2

In addition, the half-life of Alpha-galactosidase when administered at0.2 mg/kg is approximately 108 minutes. Production of GLA protein viathe “depot effect” when administering GLA mRNA-loaded lipidnanoparticles shows a substantial increase in blood residence time whencompared to direct injection of the naked recombinant protein. Asdescribed above, significant quantities of protein are present after 48hours.

The activity profile of the α-galactosidase protein produced from GLAmRNA-loaded lipid nanoparticles was measured as a function of4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-gal) metabolism. Asshown in FIG. 11, the protein produced from these nanoparticle systemsis quite active and reflective of the levels of protein available (FIG.12, Table 3). AUC comparisons of mRNA therapy-based hGLA productionversus enzyme replacement therapy (ERT) in mice and humans show a182-fold and 30-fold increase, respectively (Table 4).

TABLE 4 Comparison of C_(max) and AUC_(inf) values in Fabry patientspost-IV dosing 0.2 mg/kg of Alpha-galactosidase (pharmacological dose)with those in mice post- IV dosing Alpha-galactosidase and GLA mRNA.Test Dose C_(max) AUC_(inf) Article Descrption (mg/kg) (U/mL) (hr ·U/mL) n Fabry^(a) α-GAL Transplant 0.2 3478 3683 11 Patient ProteinDialysis 0.2 3887 3600 6 Non-ESRD^(b) 0.2 3710 4283 18 Mouse α-GALAthymic 0.04 3807 797 3 Protein nude (MM1) α-GAL Athymic 0.04 3705 602 3Protein nude (MM2) Mouse α-GAL mouse 0.95 5885 109428 6 mRNA(C_(at 6 hr))^(c) ^(a)Data were from a published paper (Gregory M.Pastores et al. Safety and Pharmacokinetics of hGLA in patients withFabry disease and end-stage renal disease. Nephrol Dial Transplant(2007) 22: 1920-1925. ^(b)non-end-stage renal disease.^(c)α-Galactosidase activity at 6 hours after dosing (the earliest timepoint tested in the study).

The ability of mRNA encapsulated lipid nanoparticles to target organswhich can act as a depot for the production of a desired protein hasbeen demonstrated. The levels of secreted protein observed have beenseveral orders of magnitude above normal physiological levels. This“depot effect” is repeatable. FIG. 12 shows again that robust proteinproduction is observed upon dosing wild type (CD-1) mice with a single30 ug dose of hGLA mRNA-loaded in C12-200-based lipid nanoparticles(Formulation 1) . In this experiment, hGLA levels were evaluated over a72 hour period. A maximum average of 4.0 ug human hGLA protein/mL serumis detected six hours post-administration. Based on a value of ˜1 ng/mLhGLA protein for normal physiological levels, hGLA MRT provides roughly4000-fold higher protein levels. As before, hGLA protein could bedetected out to 48 hr post-administration (FIG. 12).

An analysis of tissues isolated from this same experiment providedinsight into the distribution of hGLA protein in hGLA MRT-treated mice(FIG. 13). Supraphysiological levels of hGLA protein were detected inthe liver, spleen and kidneys of all mice treated with a maximumobserved between 12 and 24 hour post-administration. Detectable levelsof MRT-derived protein could be observed three days after a singleinjection of hGLA-loaded lipid nanoparticles.

In addition, the production of hGLA upon administration of hGLA mRNAloaded C12-200 nanoparticles was shown to exhibit a dose a response inthe serum (FIG. 14A), as well as in the liver (FIG. 14B).

One inherent characteristic of lipid nanoparticle-mediated mRNAreplacement therapy would be the pharmacokinetic profile of therespective protein produced. For example, ERT-based treatment of miceemploying Alpha-galactosidase results in a plasma half-life ofapproximately 100 minutes. In contrast, MRT-derived alpha-galactosidasehas a blood residence time of approximately 72 hrs with a peak time of 6hours. This allows for much greater exposure for organs to participatein possible continuous uptake of the desired protein. A comparison of PKprofiles is shown in FIG. 15 and demonstrates the stark difference inclearance rates and ultimately a major shift in area under the curve(AUC) can be achieved via MRT-based treatment.

In a separate experiment, hGLA MRT was applied to a mouse disease model,hGLA KO mice (Fabry mice). A 0.33 mg/kg dose of hGLA mRNA-loadedC12-200-based lipid nanoparticles (Formulation 1) was administered tofemale KO mice as a single, intravenous injection. Substantialquantities of MRT-derived hGLA protein were produced with a peak at 6 hr(˜560 ng/mL serum) which is approximately 600-fold higher than normalphysiological levels. Further, hGLA protein was still detectable 72 hrpost-administration (FIG. 16).

Quantification of MRT-derived GLA protein in vital organs demonstratedsubstantial accumulation as shown in FIG. 17. A comparison of observedMRT-derived hGLA protein to reported normal physiological levels thatare found in key organs is plotted (normal levels plotted as dashedlines). While levels of protein at 24 hours are higher than at 72 hourspost-administration, the levels of hGLA protein detected in the liver,kidney, spleen and hearts of the treated Fabry mice are equivalent towild type levels. For example, 3.1 ng hGLA protein/mg tissue were foundin the kidneys of treated mice 3 days after a single MRT treatment.

In a subsequent experiment, a comparison of ERT-basedAlpha-galactosidase treatment versus hGLA MRT-based treatment of maleFabry KO mice was conducted. A single, intravenous dose of 1.0 mg/kg wasgiven for each therapy and the mice were sacrificed one weekpost-administration. Serum levels of hGLA protein were monitored at 6 hrand 1 week post-injection. Liver, kidney, spleen, and heart wereanalyzed for hGLA protein accumulation one week post-administration. Inaddition to the biodistribution analyses, a measure of efficacy wasdetermined via measurement of globotrioasylceramide (Gb3) and lyso-Gb3reductions in the kidney and heart. FIG. 18 shows the serum levels ofhGLA protein after treatment of either Alpha-galactosidase or GLA mRNAloaded lipid nanoparticles (Formulation 1) in male Fabry mice. Serumsamples were analyzed at 6 hr and 1 week post-administration. A robustsignal was detected for MRT-treated mice after 6 hours, with hGLAprotein serum levels of ˜4.0 ug/mL. In contrast, there was no detectableAlpha-galactosidase remaining in the bloodstream at this time.

The Fabry mice in this experiment were sacrificed one week after theinitial injection and the organs were harvested and analyzed (liver,kidney, spleen, heart). FIG. 19 shows a comparison of human GLA proteinfound in each respective organ after either hGLA MRT orAlpha-galactosidase ERT treatment. Levels correspond to hGLA present oneweek post-administration. hGLA protein was detected in all organsanalyzed. For example, MRT-treated mice resulted in hGLA proteinaccumulation in the kidney of 2.42 ng hGLA protein/mg protein, whileAlpha-galactosidase-treated mice had only residual levels (0.37 ng/mgprotein). This corresponds to a ˜6.5-fold higher level of hGLA proteinwhen treated via hGLA MRT. Upon analysis of the heart, 11.5 ng hGLAprotein/mg protein was found for the MRT-treated cohort as compared toonly 1.0 ng/mg protein Alpha-galactosidase. This corresponds to an˜11-fold higher accumulation in the heart for hGLA MRT-treated mice overERT-based therapies.

In addition to the biodistribution analyses conducted, evaluations ofefficacy were determined via measurement of globotrioasylceramide (Gb3)and lyso-Gb3 levels in key organs. A direct comparison of Gb3 reductionafter a single, intravenous 1.0 mg/kg GLA MRT treatment as compared to aAlpha-galactosidase ERT-based therapy of an equivalent dose yielded asizeable difference in levels of Gb3 in the kidneys as well as heart.For example, Gb3 levels for GLA MRT versus Alpha-galactosidase yieldedreductions of 60.2% vs 26.8%, respectively (FIG. 20). Further, Gb3levels in the heart were reduced by 92.1% vs 66.9% for MRT andAlpha-galactosidase, respectively (FIG. 21).

A second relevant biomarker for measurement of efficacy is lyso-Gb3. GLAMRT reduced lyso-Gb3 more efficiently than Alpha-galactosidase as wellin the kidneys and heart (FIG. 20 and FIG. 21, respectively). Inparticular, MRT-treated Fabry mice demonstrated reductions of lyso-Gb3of 86.1% and 87.9% in the kidneys and heart as compared toAlpha-galactosidase-treated mice yielding a decrease of 47.8% and 61.3%,respectively.

The results with for hGLA in C12-200 based lipid nanoparticles extend toother lipid nanoparticle formulations. For example, hGLA mRNA loadedinto HGT4003 (Formulation 3) or HGT5000-based (Formulation 5) lipidnanoparticles administed as a single dose IV result in production ofhGLA at 24 hours post administration (FIG. 22). The production of hGLAexhibited a dose response. Similarly, hGLA production was observed at 6hours and 24 hours after administration of hGLA mRNA loaded intoHGT5001-based (Formulation 6) lipid nanoparticles administered as asingle dose IV. hGLA production was observed in the serum (FIG. 23A), aswell as in organs (FIG. 23B).

Overall, mRNA replacement therapy applied as a depot for proteinproduction produces large quantities of active, functionally therapeuticprotein at supraphysiological levels. This method has been demonstratedto yield a sustained circulation half-life of the desired protein andthis MRT-derived protein is highly efficacious for therapy asdemonstrated with alpha-galactosidase enzyme in Fabry mice.

1C. In Vivo Human FIX Protein Production Results

Studies were performed administering Factor IX (FIX) mRNA-loaded lipidnanoparticles in wild type mice (CD-1) and determining FIX protein thatis secreted into the bloodstream. Upon intravenous injection of a singledose of 30 ug C12-200-based (C12-200:DOPE:Chol:PEG at a ratio of40:30:25:5) FIX mRNA-loaded lipid nanoparticles (dose based onencapsulated mRNA) (Formulation 1), a robust protein production wasobserved (FIG. 24).

A pharmacokinetic analysis over 72 hours showed MRT-derived FIX proteincould be detected at all timepoints tested (FIG. 24). The peak serumconcentration was observed at 24 hr post-injection with a value of ˜3 ug(2995±738 ng/mL) FIX protein/mL serum. This represents anothersuccessful example of the depot effect.

1D. In Vivo Human A1AT Protein Production Results

Studies were performed administering alpha-1-antitrypsin (A1AT)mRNA-loaded lipid nanoparticles in wild type mice (CD-1) and determiningA1AT protein that is secreted into the bloodstream. Upon intravenousinjection of a single dose of 30 ug C12-200-based A1AT mRNA-loaded lipidnanoparticles (dose based on encapsulated mRNA) (Formulation 1), arobust protein production was observed (FIG. 25).

As depicted in FIG. 25, detectable levels of human A1AT protein derivedfrom A1AT MRT could be observed over a 24 hour time periodpost-administration. A maximum serum level of ˜48 ug A1AT protein/mLserum was detected 12 hours after injection.

Example 2: Protein Production Depot Via Pulmonary Delivery ofPolynucleotide Compositions Injection Protocol

All studies were performed using female CD-1 or BALB/C mice ofapproximately 7-10 weeks of age at the beginning of each experiment.Test articles were introduced via a single intratracheal aerosolizedadministration. Mice were sacrificed and perfused with saline at thedesignated time points. The lungs of each mouse were harvested,apportioned into two parts, and stored in either 10% neutral bufferedformalin or snap-frozen and stored at −80° C. for analysis. Serum wasisolated as described in Example 1. EPO ELISA: as described in Example1.

Results

The depot effect can be achieved via pulmonary delivery (e.g.intranasal, intratracheal, nebulization). Measurement of the desiredexogenous-based protein derived from messenger RNA delivered viananoparticle systems was achieved and quantified.

The production of human EPO protein via hEPO mRNA-loaded lipidnanoparticles was tested in CD-1 mice via a single intratrachealadministration (MicroSprayer®). Several formulations were tested usingvarious cationic lipids (Formulations 1, 5, 6). All formulationsresulted in high encapsulation of human EPO mRNA. Upon administration,animals were sacrificed six hours post-administration and the lungs aswell as serum were harvested.

Human EPO protein was detected at the site of administration (lungs)upon treatment via aerosol delivery. Analysis of the serum six hourspost-administration showed detectable amounts of hEPO protein incirculation. These data (shown in FIG. 26) demonstrate the ability ofthe lung to act as a “depot” for the production (and secretion) of hEPOprotein.

1-40. (canceled)
 41. A method for delivery of messenger RNA (mRNA) forin vivo production of a factor IX (FIX) protein, comprisingadministering, to a human, a composition comprising an mRNA that encodesthe FIX protein wherein the mRNA is encapsulated within a lipidnanoparticle, wherein the administering of the composition results inexpression of the FIX protein encoded by the mRNA that is detectable inserum at least 72 hours after administration, and wherein the lipidnanoparticle comprises one or more PEG-modified lipids.
 42. The methodof claim 41, wherein the mRNA comprises a 5′ untranslated region. 43.The method of claim 41, wherein the mRNA comprises a 3′ untranslatedregion.
 44. The method of claim 43, wherein the mRNA comprises a capstructure.
 45. The method of claim 44, wherein the mRNA comprises a polyA tail.
 46. The method of claim 45, wherein the lipid nanoparticlecomprises one or more cationic lipids.
 47. The method of claim 46,wherein the lipid nanoparticle comprises one or more non-cationiclipids.
 48. The method of claim 47, wherein the mRNA is unmodified. 49.The method of claim 48, wherein the composition is a reconstitutedlyophilized composition.
 50. The method of claim 47, wherein themodification comprises a modified nucleotide.
 51. The method of claim50, wherein the modified nucleotide is pseudouridine.
 52. The method ofclaim 51, wherein the composition is a reconstituted lyophilizedcomposition.
 53. The method of claim 48, wherein the composition isadministered by intravenous injection.
 54. The method of claim 51,wherein the composition is administered by intravenous injection. 55.The method of claim 48, wherein the composition is administered byintramuscular injection.
 56. The method of claim 51, wherein thecomposition is administered by intramuscular injection.
 57. The methodof claim 48, wherein the lipid nanoparticle has a size of less thanabout 100 nm.
 58. The method of claim 51, wherein the lipid nanoparticlehas a size of less than about 100 nm.
 59. The method of claim 48,wherein the one or more PEG-modified lipids constitute from 0.1% to 20%of the total lipids by a molar ratio.
 60. The method of claim 51,wherein the one or more PEG-modified lipids constitute from 0.1% to 20%of the total lipids by a molar ratio.
 61. The method of claim 48,wherein the poly A tail comprises at least 90 nucleotides.
 62. Themethod of claim 51, wherein the poly A tail comprises at least 500nucleotides.